Riboswitch reporter assays

Riboswitch-reporter constructs, integrating riboswitch representatives from either C. sp Maddingley or C. lundense DSM 17049, were prepared as synthetic oligonucleotides, amplified by PCR and cloned into vector pDG1661 upstream of the E. coli lacZ gene as described previously (Sudarsan et al., 2003; Nelson et al., 2017). Transcription initiation of the constructs is driven by the B. subtilis lysC gene promoter. The resulting WT and mutant reporter constructs were integrated into the amyE locus of WT (1A1 strain 168 Δtrp) or thiamin biosynthetic knockout strains (ΔthiS, ΔthiE or ΔthiD) as indicated. The resulting transformed strains were verified as previously described (Sherlock et al., 2018).

The thiD gene construct was generated by amplifying this gene from B. subtilis genomic DNA by PCR and inserted into the StuI site of a modified pDG148 vector using ligation-independent cloning as described previously (Joseph et al., 2001). The lacI gene in this vector has been mutated so that the thiD gene is expected to give constitutive expression. The resulting protein expression vector was then transformed into B. subtilis strains containing WT or mutant riboswitch reporter constructs, as indicated for each experiment.

Riboswitch-reporter assays were performed by inoculating various B. subtilis strains into Lysogeny Broth (LB) with appropriate antibiotics and growing overnight at 37°C. For liquid-culture reporter assays with thiamin biosynthetic knock-out strains, overnight cultures grown in LB were then diluted 1/20 into Spizizen glucose minimal medium (GMM) (Anagnostopoulos and Spizizen, 1961) and grown overnight at 37°C. The residual thiamin from LB is sufficient for growth in GMM over the duration of the assay. For riboswitch reporter experiments with ThiD-producing strains, bacteria were diluted directly into LB and grown overnight with or without supplementation with HMP. Liquid media (LB or GMM) was supplemented with X-gal (200 μg mL⁻¹) to allow visual detection of reporter gene expression. Similarly, reporter expression analysis using 4-methylumbelliferyl β-D-galactopyranoside was conducted as described previously (Nelson et al., 2015; Atilho et al., 2019) to establish fluorescence units.